HS project

Combined dataset

This file is used to generate a dataset containing all individual datasets, without melanocytes.

library(dplyr)
library(patchwork)
library(ggplot2)
library(ComplexHeatmap)

.libPaths()

## [1] "/usr/local/lib/R/library"

Preparation

In this section, we set the global settings of the analysis. We will store data there :

save_name = "hs_hd"
out_dir = "."
n_threads = 5 # for tSNE

We load the sample information :

sample_info = readRDS(paste0(out_dir, "/../1_metadata/hs_hd_sample_info.rds"))
project_names_oi = sample_info$project_name

graphics::pie(rep(1, nrow(sample_info)),
              col = sample_info$color,
              labels = sample_info$project_name)

Here are custom colors for each cell type :

color_markers = readRDS(paste0(out_dir, "/../1_metadata/hs_hd_color_markers.rds"))

data.frame(cell_type = names(color_markers),
           color = unlist(color_markers)) %>%
  ggplot2::ggplot(., aes(x = cell_type, y = 0, fill = cell_type)) +
  ggplot2::geom_point(pch = 21, size = 5) +
  ggplot2::scale_fill_manual(values = unlist(color_markers), breaks = names(color_markers)) +
  ggplot2::theme_classic() +
  ggplot2::theme(legend.position = "none",
                 axis.line = element_blank(),
                 axis.title = element_blank(),
                 axis.ticks = element_blank(),
                 axis.text.y = element_blank())

We load the markers and specific colors for each cell type :

cell_markers = readRDS(paste0(out_dir, "/../1_metadata/hs_hd_cell_markers.rds"))
cell_markers = lapply(cell_markers, FUN = toupper)
lengths(cell_markers)

##     T_CD4     T_CD8  mac_CPVL mac_TREM2        FC        CO        ME       IBL 
##        13        13         7        10        10        15        10        15 
##        IF      mORS    HFSC_1    HFSC_2      mela 
##        22        14        17        18        10

We load markers to display on the dotplot :

dotplot_markers = readRDS(paste0(out_dir, "/../1_metadata/hs_hd_dotplot_markers.rds"))
dotplot_markers = lapply(dotplot_markers, FUN = toupper)
dotplot_markers

## $T_CD4
## [1] "CD3E" "CD4" 
## 
## $T_CD8
## [1] "CD3E" "CD8A"
## 
## $mac_CPVL
## [1] "AIF1" "CPVL"
## 
## $mac_TREM2
## [1] "AIF1"  "TREM2"
## 
## $FC
## [1] "CD3E" "CD8A"
## 
## $CO
## [1] "KRT35" "KRT85"
## 
## $ME
## [1] "BAMBI"  "SLC7A8"
## 
## $IBL
## [1] "KRT16" "KRT6B"
## 
## $IF
## [1] "AQP3"   "S100A8"
## 
## $mORS
## [1] "IMPA2" "KRT6A"
## 
## $HFSC_1
## [1] "DIO2"  "TGFB2"
## 
## $HFSC_2
## [1] "DIO2"    "BHLHE41"
## 
## $mela
## [1] "DCT"   "MLANA"

Make hs_hd dataset

Individual datasets

For each sample, we :

• load individual dataset
• look at cell annotation

We load individual datasets :

sobj_list = lapply(project_names_oi, FUN = function(one_project_name) {
  subsobj = readRDS(paste0(out_dir, "/../2_individual/datasets/",
                           one_project_name, "_sobj_filtered.rds"))
  return(subsobj)
})
names(sobj_list) = project_names_oi

lapply(sobj_list, FUN = dim) %>%
  do.call(rbind, .) %>%
  rbind(., colSums(.))

##           [,1]  [,2]
## 2021_31  27955  1043
## 2021_36  27955   602
## 2021_41  27955  2256
## 2022_03  27955  3977
## 2022_14  27955  2588
## 2022_01  27955  1213
## 2022_02  27955  2286
##         195685 13965

We represent cells in the tSNE :

name2D = "RNA_pca_20_tsne"

We look at cell type annotation for each dataset :

plot_list = lapply(sobj_list, FUN = function(one_sobj) {
  mytitle = as.character(unique(one_sobj$project_name))
  mysubtitle = ncol(one_sobj)
  
  p = Seurat::DimPlot(one_sobj, group.by = "cell_type",
                      reduction = name2D) +
    ggplot2::scale_color_manual(values = color_markers,
                                breaks = names(color_markers),
                                name = "Cell Type") +
    ggplot2::labs(title = mytitle,
                  subtitle = paste0(mysubtitle, " cells")) +
    ggplot2::theme(aspect.ratio = 1,
                   plot.title = element_text(hjust = 0.5),
                   plot.subtitle = element_text(hjust = 0.5)) +
    Seurat::NoAxes()
  
  return(p)
})

plot_list[[length(plot_list) + 1]] = patchwork::guide_area()

patchwork::wrap_plots(plot_list, ncol = 4) +
  patchwork::plot_layout(guides = "collect") &
  ggplot2::theme(legend.position = "right")

and clustering :

plot_list = lapply(sobj_list, FUN = function(one_sobj) {
  mytitle = as.character(unique(one_sobj$project_name))
  mysubtitle = ncol(one_sobj)
  
  p = Seurat::DimPlot(one_sobj, group.by = "seurat_clusters",
                      reduction = name2D, label = TRUE) +
    ggplot2::labs(title = mytitle,
                  subtitle = paste0(mysubtitle, " cells")) +
    ggplot2::theme(aspect.ratio = 1,
                   plot.title = element_text(hjust = 0.5),
                   plot.subtitle = element_text(hjust = 0.5)) +
    Seurat::NoAxes() + Seurat::NoLegend()
  
  return(p)
})

patchwork::wrap_plots(plot_list, ncol = 4)

Melanocytes remomal

For each individual dataset, we remove melanocytes. First, we smooth cell type annotation at a cluster level :

sobj_list = lapply(sobj_list, FUN = function(one_sobj) {
  cluster_type = table(one_sobj$cell_type, one_sobj$seurat_clusters) %>%
    prop.table(., margin = 2) %>%
    apply(., 2, which.max)
  cluster_type = setNames(nm = names(cluster_type),
                          levels(one_sobj$cell_type)[cluster_type])
  
  one_sobj$cluster_type = cluster_type[one_sobj$seurat_clusters]
  
  ## Output
  return(one_sobj)
})

To locate melanocytes, we look at their score, cell type annotation, and clustering.

plot_list = lapply(sobj_list, FUN = function(one_sobj) {
  project_name = as.character(unique(one_sobj$project_name))
  plot_sublist = list()
  
  # Score
  plot_sublist[[1]] = Seurat::FeaturePlot(one_sobj, reduction = name2D,
                                          features = "score_mela") +
    ggplot2::labs(title = project_name,
                  subtitle = "Melanocytes score") +
    Seurat::NoAxes() +
    ggplot2::scale_color_gradientn(colors = aquarius:::color_gene) +
    ggplot2::theme(aspect.ratio = 1,
                   plot.subtitle = element_text(hjust = 0.5))
  
  # Cell type
  plot_sublist[[2]] = Seurat::DimPlot(one_sobj,
                                      reduction = name2D,
                                      group.by = "cell_type",
                                      order = "melanocytes") +
    ggplot2::scale_color_manual(values = c("purple", rep("gray92", length(color_markers) - 1)),
                                breaks = c("mela", setdiff(names(color_markers), "mela"))) +
    ggplot2::labs(title = "Cell type annotation",
                  subtitle = paste0(sum(one_sobj$cell_type == "mela"),
                                    " melanocytes")) +
    Seurat::NoAxes() + Seurat::NoLegend() +
    ggplot2::theme(aspect.ratio = 1,
                   plot.title = element_text(hjust = 0.5),
                   plot.subtitle = element_text(hjust = 0.5))
  
  # Clusters
  plot_sublist[[3]] = Seurat::DimPlot(one_sobj,
                                      reduction = name2D,
                                      group.by = "seurat_clusters",
                                      label = TRUE) +
    ggplot2::labs(title = "Clusters") +
    Seurat::NoAxes() + Seurat::NoLegend() +
    ggplot2::theme(aspect.ratio = 1,
                   plot.title = element_text(hjust = 0.5),
                   plot.subtitle = element_text(hjust = 0.5))
  
  # Cluster type
  plot_sublist[[4]] = Seurat::DimPlot(one_sobj,
                                      reduction = name2D,
                                      group.by = "cluster_type") +
    ggplot2::scale_color_manual(values = c("purple", rep("gray92", length(color_markers) - 1)),
                                breaks = c("mela", setdiff(names(color_markers), "mela"))) +
    ggplot2::labs(title = "Cluster annotation",
                  subtitle = paste0(sum(one_sobj$cluster_type == "mela"),
                                    " melanocytes")) +
    Seurat::NoAxes() + Seurat::NoLegend() +
    ggplot2::theme(aspect.ratio = 1,
                   plot.title = element_text(hjust = 0.5),
                   plot.subtitle = element_text(hjust = 0.5))
  
  return(plot_sublist)
}) %>% unlist(., recursive = FALSE)

patchwork::wrap_plots(plot_list, ncol = 4)

We remove melanocytes based on cluster annotation :

sobj_list = lapply(sobj_list, FUN = function(one_sobj) {
  one_sobj$is_of_interest = (one_sobj$cluster_type != "mela")
  
  if (sum(one_sobj$is_of_interest) > 0) {
    one_sobj = subset(one_sobj, is_of_interest == TRUE)
  } else {
    one_sobj = NA
  }
  
  one_sobj$is_of_interest = NULL
  return(one_sobj)
})

lapply(sobj_list, FUN = dim) %>%
  do.call(rbind, .) %>%
  rbind(., colSums(.))

##           [,1]  [,2]
## 2021_31  27955   885
## 2021_36  27955   528
## 2021_41  27955  1982
## 2022_03  27955  3457
## 2022_14  27955  2422
## 2022_01  27955   835
## 2022_02  27955  2002
##         195685 12111

Re-annotation

We remove melanocytes from annotation :

cell_markers = cell_markers[names(cell_markers) != "mela"]
color_markers = color_markers[names(color_markers) != "mela"]
dotplot_markers = dotplot_markers[names(dotplot_markers) != "mela"]

We re-annote cells for cell type, since melanocytes have been removed :

sobj_list = lapply(sobj_list, FUN = function(one_sobj) {
  # Remove old annotation
  one_sobj@meta.data[, grep(colnames(one_sobj@meta.data), pattern = "score", value = TRUE)] = NULL
  
  # Re-annot
  one_sobj = aquarius::cell_annot_custom(one_sobj,
                                         newname = "cell_type",
                                         markers = cell_markers,
                                         use_negative = TRUE,
                                         add_score = FALSE,
                                         verbose = TRUE)
  
  # Set factor levels
  one_sobj$cell_type = factor(one_sobj$cell_type, levels = names(cell_markers))
  
  return(one_sobj)
})

Combined dataset

We combine all datasets :

sobj = base::merge(sobj_list[[1]],
                   y = sobj_list[c(2:length(sobj_list))],
                   add.cell.ids = names(sobj_list))
sobj

## An object of class Seurat 
## 27955 features across 12111 samples within 1 assay 
## Active assay: RNA (27955 features, 0 variable features)

We add again the correspondence between gene names and gene ID. Since all datasets have been aligned using the same transcriptome, we take the correspondence from one individual dataset.

sobj@assays$RNA@meta.features = sobj_list[[1]]@assays$RNA@meta.features[, c("Ensembl_ID", "gene_name")]

head(sobj@assays$RNA@meta.features)

##                  Ensembl_ID   gene_name
## MIR1302-2HG ENSG00000243485 MIR1302-2HG
## FAM138A     ENSG00000237613     FAM138A
## OR4F5       ENSG00000186092       OR4F5
## AL627309.1  ENSG00000238009  AL627309.1
## AL627309.3  ENSG00000239945  AL627309.3
## AL627309.4  ENSG00000241599  AL627309.4

We remove the list of objects :

rm(sobj_list)

We keep a subset of meta.data and reset levels :

sobj@meta.data = sobj@meta.data[, c("orig.ident", "nCount_RNA", "nFeature_RNA", "log_nCount_RNA",
                                    "project_name", "sample_identifier", "sample_type",
                                    "Seurat.Phase", "cyclone.Phase", "percent.mt", "percent.rb",
                                    "cell_type")]

sobj$orig.ident = factor(sobj$orig.ident, levels = levels(sample_info$project_name))
sobj$project_name = factor(sobj$project_name, levels = levels(sample_info$project_name))
sobj$sample_identifier = factor(sobj$sample_identifier, levels = levels(sample_info$sample_identifier))
sobj$sample_type = factor(sobj$sample_type, levels = levels(sample_info$sample_type))
sobj$cell_type = factor(sobj$cell_type, levels = names(color_markers))

summary(sobj@meta.data)

##    orig.ident     nCount_RNA      nFeature_RNA  log_nCount_RNA    project_name 
##  2021_31: 885   Min.   :   658   Min.   : 500   Min.   : 6.489   2021_31: 885  
##  2021_36: 528   1st Qu.:  4398   1st Qu.:1574   1st Qu.: 8.389   2021_36: 528  
##  2021_41:1982   Median : 11674   Median :3043   Median : 9.365   2021_41:1982  
##  2022_03:3457   Mean   : 15151   Mean   :3082   Mean   : 9.181   2022_03:3457  
##  2022_14:2422   3rd Qu.: 22206   3rd Qu.:4394   3rd Qu.:10.008   2022_14:2422  
##  2022_01: 835   Max.   :139803   Max.   :7942   Max.   :11.848   2022_01: 835  
##  2022_02:2002                                                    2022_02:2002  
##  sample_identifier sample_type Seurat.Phase       cyclone.Phase     
##  HS_1: 885         HS:9274     Length:12111       Length:12111      
##  HS_2: 528         HD:2837     Class :character   Class :character  
##  HS_3:1982                     Mode  :character   Mode  :character  
##  HS_4:3457                                                          
##  HS_5:2422                                                          
##  HD_1: 835                                                          
##  HD_2:2002                                                          
##    percent.mt       percent.rb        cell_type   
##  Min.   : 0.000   Min.   : 0.4948   IF     :2393  
##  1st Qu.: 2.902   1st Qu.:20.0623   CO     :1774  
##  Median : 4.710   Median :24.7964   IBL    :1721  
##  Mean   : 5.025   Mean   :24.1542   FC     :1157  
##  3rd Qu.: 6.512   3rd Qu.:29.2363   T_CD4  : 897  
##  Max.   :19.954   Max.   :46.0169   HFSC_1 : 790  
##                                     (Other):3379

Processing

We remove genes that are expressed in less than 5 cells :

sobj = aquarius::filter_features(sobj, min_cells = 5)

## [1] 27955 12111
## [1] 20003 12111

sobj

## An object of class Seurat 
## 20003 features across 12111 samples within 1 assay 
## Active assay: RNA (20003 features, 0 variable features)

Metadata

How many cells by sample ?

table(sobj$project_name)

## 
## 2021_31 2021_36 2021_41 2022_03 2022_14 2022_01 2022_02 
##     885     528    1982    3457    2422     835    2002

We represent this information as a barplot :

aquarius::plot_barplot(df = table(sobj$project_name,
                                  sobj$cell_type) %>%
                         as.data.frame.table() %>%
                         `colnames<-`(c("Sample", "Cell Type", "Number")),
                       x = "Sample", y = "Number", fill = "Cell Type",
                       position = position_fill()) +
  ggplot2::scale_fill_manual(values = unlist(color_markers),
                             breaks = names(color_markers),
                             name = "Cell Type")

This is the same barplot with another position :

aquarius::plot_barplot(df = table(sobj$project_name,
                                  sobj$cell_type) %>%
                         as.data.frame.table() %>%
                         `colnames<-`(c("Sample", "Cell Type", "Number")),
                       x = "Sample", y = "Number", fill = "Cell Type",
                       position = position_stack()) +
  ggplot2::scale_fill_manual(values = unlist(color_markers),
                             breaks = names(color_markers),
                             name = "Cell Type")

Projection

We normalize the count matrix for remaining cells and select highly variable features :

sobj = Seurat::NormalizeData(sobj,
                             normalization.method = "LogNormalize")
sobj = Seurat::FindVariableFeatures(sobj, nfeatures = 2000)
sobj = Seurat::ScaleData(sobj)

sobj

## An object of class Seurat 
## 20003 features across 12111 samples within 1 assay 
## Active assay: RNA (20003 features, 2000 variable features)

We perform a PCA :

sobj = Seurat::RunPCA(sobj,
                      assay = "RNA",
                      reduction.name = "RNA_pca",
                      npcs = 100,
                      seed.use = 1337L)
sobj

## An object of class Seurat 
## 20003 features across 12111 samples within 1 assay 
## Active assay: RNA (20003 features, 2000 variable features)
##  1 dimensional reduction calculated: RNA_pca

We choose the number of dimensions such that they summarize 60 % of the variability :

stdev = sobj@reductions[["RNA_pca"]]@stdev
stdev_prop = cumsum(stdev)/sum(stdev)
ndims = which(stdev_prop > 0.60)[1]
ndims

## [1] 38

We can visualize this on the elbow plot :

elbow_p = Seurat::ElbowPlot(sobj, ndims = 100, reduction = "RNA_pca") +
  ggplot2::geom_point(x = ndims, y = stdev[ndims], col = "red")
x_text = ggplot_build(elbow_p)$layout$panel_params[[1]]$x$get_labels() %>% as.numeric()
elbow_p = elbow_p +
  ggplot2::scale_x_continuous(breaks = sort(c(x_text, ndims)), limits = c(0, 100))
x_color = ifelse(ggplot_build(elbow_p)$layout$panel_params[[1]]$x$get_labels() %>%
                   as.numeric() %>% round(., 2) == round(ndims, 2), "red", "black")
elbow_p = elbow_p +
  ggplot2::theme_classic() +
  ggplot2::theme(axis.text.x = element_text(color = x_color))

elbow_p

We generate a tSNE and a UMAP with 38 principal components :

sobj = Seurat::RunTSNE(sobj,
                       reduction = "RNA_pca",
                       dims = 1:ndims,
                       seed.use = 1337L,
                       num_threads = n_threads, # Rtsne::Rtsne option
                       reduction.name = paste0("RNA_pca_", ndims, "_tsne"))

sobj = Seurat::RunUMAP(sobj,
                       reduction = "RNA_pca",
                       dims = 1:ndims,
                       seed.use = 1337L,
                       reduction.name = paste0("RNA_pca_", ndims, "_umap"))

(Time to run : 37.63 s)

We can visualize the two representations :

tsne = Seurat::DimPlot(sobj, group.by = "project_name",
                       reduction = paste0("RNA_pca_", ndims, "_tsne")) +
  ggplot2::scale_color_manual(values = sample_info$color,
                              breaks = sample_info$project_name) +
  Seurat::NoAxes() + ggplot2::ggtitle("PCA - tSNE") +
  ggplot2::theme(aspect.ratio = 1,
                 plot.title = element_text(hjust = 0.5),
                 legend.position = "none")

umap = Seurat::DimPlot(sobj, group.by = "project_name",
                       reduction = paste0("RNA_pca_", ndims, "_umap")) +
  ggplot2::scale_color_manual(values = sample_info$color,
                              breaks = sample_info$project_name) +
  Seurat::NoAxes() + ggplot2::ggtitle("PCA - UMAP") +
  ggplot2::theme(aspect.ratio = 1,
                 plot.title = element_text(hjust = 0.5))

tsne | umap

Batch-effect correction

We remove sample specific effect on the pca using Harmony :

`%||%` = function(lhs, rhs) {
  if (!is.null(x = lhs)) {
    return(lhs)
  } else {
    return(rhs)
  }
}

set.seed(1337L)
sobj = harmony::RunHarmony(object = sobj,
                           group.by.vars = "project_name",
                           plot_convergence = TRUE,
                           reduction = "RNA_pca",
                           assay.use = "RNA",
                           reduction.save = "harmony",
                           max.iter.harmony = 50,
                           project.dim = FALSE)

(Time to run : 63.26 s)

From this batch-effect removed projection, we generate a tSNE and a UMAP.

sobj = Seurat::RunUMAP(sobj, 
                       seed.use = 1337L,
                       dims = 1:ndims,
                       reduction = "harmony",
                       reduction.name = paste0("harmony_", ndims, "_umap"),
                       reduction.key = paste0("harmony_", ndims, "umap_"))

sobj = Seurat::RunTSNE(sobj,
                       dims = 1:ndims,
                       seed.use = 1337L,
                       num_threads = n_threads, # Rtsne::Rtsne option
                       reduction = "harmony",
                       reduction.name = paste0("harmony_", ndims, "_tsne"),
                       reduction.key = paste0("harmony", ndims, "tsne_"))

(Time to run : 39.48 s)

We visualize the corrected projections :

tsne = Seurat::DimPlot(sobj, group.by = "project_name",
                       reduction = paste0("harmony_", ndims, "_tsne")) +
  ggplot2::scale_color_manual(values = sample_info$color,
                              breaks = sample_info$project_name) +
  Seurat::NoAxes() + ggplot2::ggtitle("PCA - harmony - tSNE") +
  ggplot2::theme(aspect.ratio = 1,
                 plot.title = element_text(hjust = 0.5),
                 legend.position = "none")

umap = Seurat::DimPlot(sobj, group.by = "project_name",
                       reduction = paste0("harmony_", ndims, "_umap")) +
  ggplot2::scale_color_manual(values = sample_info$color,
                              breaks = sample_info$project_name) +
  Seurat::NoAxes() + ggplot2::ggtitle("PCA - harmony - UMAP") +
  ggplot2::theme(aspect.ratio = 1,
                 plot.title = element_text(hjust = 0.5))

tsne | umap

We will keep the tSNE from harmony :

reduction = "harmony"
name2D = paste0("harmony_", ndims, "_tsne")

Clustering

We generate a clustering :

sobj = Seurat::FindNeighbors(sobj, reduction = reduction, dims = 1:ndims)
sobj = Seurat::FindClusters(sobj, resolution = 1)

## Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck
## 
## Number of nodes: 12111
## Number of edges: 475472
## 
## Running Louvain algorithm...
## Maximum modularity in 10 random starts: 0.9021
## Number of communities: 27
## Elapsed time: 1 seconds

clusters_plot = Seurat::DimPlot(sobj, reduction = name2D, label = TRUE) +
  Seurat::NoAxes() + Seurat::NoLegend() +
  ggplot2::labs(title = "Clusters ID") +
  ggplot2::theme(aspect.ratio = 1,
                 plot.title = element_text(hjust = 0.5))
clusters_plot

Visualization

We represent the 4 quality metrics :

plot_list = Seurat::FeaturePlot(sobj, reduction = name2D,
                                combine = FALSE, pt.size = 0.25,
                                features = c("percent.mt", "percent.rb", "log_nCount_RNA", "nFeature_RNA"))
plot_list = lapply(plot_list, FUN = function(one_plot) {
  one_plot +
    Seurat::NoAxes() +
    ggplot2::scale_color_gradientn(colors = aquarius:::color_gene) +
    ggplot2::theme(aspect.ratio = 1)
})

patchwork::wrap_plots(plot_list, nrow = 1)

Clusters

We can represent clusters, split by sample of origin :

plot_list = aquarius::plot_split_dimred(sobj,
                                        reduction = name2D,
                                        split_by = "project_name",
                                        group_by = "seurat_clusters",
                                        split_color = setNames(sample_info$color,
                                                               nm = sample_info$project_name),
                                        group_color = aquarius::gg_color_hue(length(levels(sobj$seurat_clusters))))

plot_list[[length(plot_list) + 1]] = clusters_plot +
  ggplot2::labs(title = "Cluster ID") &
  ggplot2::theme(plot.title = element_text(hjust = 0.5, size = 15))

patchwork::wrap_plots(plot_list, ncol = 4) &
  Seurat::NoLegend()

We make a heatmap to compare the representativness of cells for each sample, within each cluster :

cluster_by_sample = table(sobj$sample_identifier,
                          sobj@meta.data[, "seurat_clusters"]) %>%
  prop.table(margin = 1) %>%
  as.matrix()

## Representative markers
cluster_markers = c("PTPRC", "CD3E", "AIF1", "MSX2", "DIO2", "GPX2", "KRT16")
ht_annot = Seurat::GetAssayData(sobj, assay = "RNA", slot = "data")[cluster_markers, ] %>%
  Matrix::t() %>% as.data.frame()
ht_annot$clusters = sobj@meta.data[, "seurat_clusters"]
ht_annot = ht_annot %>%
  dplyr::group_by(clusters) %>%
  dplyr::summarise_all(funs('mean' = mean)) %>%
  as.data.frame() %>%
  dplyr::select(-clusters) %>%
  `colnames<-`(c(cluster_markers))

color_fun = function(one_gene) {
  gene_range = range(ht_annot[, one_gene])
  gene_palette = circlize::colorRamp2(colors = c("#FFFFFF", aquarius::color_gene[-1]),
                                      breaks = seq(from = gene_range[1], to = gene_range[2],
                                                   length.out = length(aquarius::color_gene)))
  return(gene_palette)
}

## Bottom annotation : average gene expression
ha_bottom = ComplexHeatmap::HeatmapAnnotation(df = ht_annot,
                                              which = "column",
                                              show_legend = FALSE,
                                              col = setNames(nm = cluster_markers,
                                                             lapply(cluster_markers, FUN = color_fun)),
                                              annotation_name_side = "left")

## Right annotation : number of cells by dataset
ht_annot = table(sobj$sample_identifier) %>%
  as.data.frame.table() %>%
  `colnames<-`(c("sample_identifier", "nb_cells")) %>%
  `rownames<-`(.$sample_identifier) %>%
  dplyr::select(-sample_identifier)

ha_right = ComplexHeatmap::HeatmapAnnotation(
  df = ht_annot,
  which = "row",
  show_legend = TRUE,
  annotation_name_side = "top",
  col = list(nb_cells  = circlize::colorRamp2(colors = RColorBrewer::brewer.pal(name = "Greys", n = 9),
                                              breaks = seq(from = range(ht_annot$nb_cells)[1],
                                                           to = range(ht_annot$nb_cells)[2],
                                                           length.out = 9))))

## Top annotation : main cell type in this cluster
ht_annot = table(sobj$sample_identifier,
                 sobj@meta.data[, "seurat_clusters"]) %>%
  prop.table(margin = 1) %>%
  as.matrix()

ht_annot = table(sobj$cell_type,
                 sobj$seurat_clusters) %>%
  prop.table(., margin = 2) %>%
  apply(., 2, which.max)
ht_annot = data.frame(row.names = names(ht_annot),
                      cell_type = names(color_markers)[ht_annot])

ha_top = ComplexHeatmap::HeatmapAnnotation(
  df = ht_annot,
  which = "column",
  show_legend = TRUE,
  annotation_name_side = "left",
  col = list(cell_type = color_markers))

## Assemble heatmap
ht = ComplexHeatmap::Heatmap(cluster_by_sample,
                             heatmap_legend_param = list(title = "Proportion",
                                                         col = c("#2166AC", "#F7F7F7", "#B2182B")),
                             bottom_annotation = ha_bottom,
                             right_annotation = ha_right,
                             top_annotation = ha_top,
                             cluster_rows = TRUE,
                             cluster_columns = TRUE,
                             row_title = "Sample",
                             row_names_gp = grid::gpar(names = sample_info$sample_identifier,
                                                       col = sample_info$color,
                                                       fontface = "bold"),
                             column_title = "Cluster",
                             row_names_side = "left",
                             column_names_side = "top",
                             column_names_rot = 0)

## Draw !
ComplexHeatmap::draw(ht, merge_legends = TRUE)

Cell type

We visualize cell type :

plot_list = lapply((c(paste0("RNA_pca_", ndims, "_tsne"),
                      paste0("RNA_pca_", ndims, "_umap"),
                      paste0("harmony_", ndims, "_tsne"),
                      paste0("harmony_", ndims, "_umap"))),
                   FUN = function(one_red) {
                     Seurat::DimPlot(sobj, group.by = "cell_type",
                                     reduction = one_red,
                                     cols = color_markers) +
                       Seurat::NoAxes() + ggplot2::ggtitle(one_red) +
                       ggplot2::theme(aspect.ratio = 1,
                                      plot.title = element_text(hjust = 0.5))
                   })

patchwork::wrap_plots(plot_list, nrow = 2) +
  patchwork::plot_layout(guides = "collect")

We make a representation split by origin to show cell types :

plot_list = aquarius::plot_split_dimred(sobj,
                                        reduction = name2D,
                                        split_by = "project_name",
                                        split_color = setNames(sample_info$color,
                                                               nm = sample_info$project_name),
                                        group_by = "cell_type",
                                        group_color = color_markers)

plot_list[[length(plot_list) + 1]] = patchwork::guide_area()

patchwork::wrap_plots(plot_list, ncol = 4) +
  patchwork::plot_layout(guides = "collect") &
  ggplot2::theme(legend.position = "right")

Cell cycle

We visualize cell cycle annotation, and BIRC5 and TOP2A expression levels :

plot_list = list()

# Seurat
plot_list[[1]] = Seurat::DimPlot(sobj, group.by = "Seurat.Phase",
                                 reduction = name2D) +
  Seurat::NoAxes() + ggplot2::labs(title = "Seurat annotation") +
  ggplot2::theme(aspect.ratio = 1,
                 plot.title = element_text(hjust = 0.5))

# cyclone
plot_list[[2]] = Seurat::DimPlot(sobj, group.by = "cyclone.Phase",
                                 reduction = name2D) +
  Seurat::NoAxes() + ggplot2::labs(title = "cyclone annotation") +
  ggplot2::theme(aspect.ratio = 1,
                 plot.title = element_text(hjust = 0.5))

# BIRC5
plot_list[[3]] = Seurat::FeaturePlot(sobj, features = "BIRC5",
                                     reduction = name2D) +
  ggplot2::scale_color_gradientn(colors = aquarius::color_gene) +
  Seurat::NoAxes() +
  ggplot2::theme(aspect.ratio = 1,
                 plot.title = element_text(hjust = 0.5))

# TOP2A
plot_list[[4]] = Seurat::FeaturePlot(sobj, features = "TOP2A",
                                     reduction = name2D) +
  ggplot2::scale_color_gradientn(colors = aquarius::color_gene) +
  Seurat::NoAxes() +
  ggplot2::theme(aspect.ratio = 1,
                 plot.title = element_text(hjust = 0.5))

patchwork::wrap_plots(plot_list, ncol = 2)

Save

We save the Seurat object :

saveRDS(sobj, file = paste0(out_dir, "/", save_name, "_sobj.rds"))

R Session

show

## R version 3.6.3 (2020-02-29)
## Platform: x86_64-pc-linux-gnu (64-bit)
## Running under: Ubuntu 20.04.6 LTS
## 
## Matrix products: default
## BLAS:   /usr/local/lib/R/lib/libRblas.so
## LAPACK: /usr/local/lib/R/lib/libRlapack.so
## 
## locale:
## [1] C
## 
## attached base packages:
## [1] grid      stats     graphics  grDevices utils     datasets  methods  
## [8] base     
## 
## other attached packages:
## [1] ComplexHeatmap_2.14.0 ggplot2_3.3.5         patchwork_1.1.2      
## [4] dplyr_1.0.7          
## 
## loaded via a namespace (and not attached):
##   [1] softImpute_1.4              graphlayouts_0.7.0         
##   [3] pbapply_1.4-2               lattice_0.20-41            
##   [5] haven_2.3.1                 vctrs_0.3.8                
##   [7] usethis_2.0.1               dynwrap_1.2.1              
##   [9] blob_1.2.1                  survival_3.2-13            
##  [11] prodlim_2019.11.13          dynutils_1.0.5             
##  [13] later_1.3.0                 DBI_1.1.1                  
##  [15] R.utils_2.11.0              SingleCellExperiment_1.8.0 
##  [17] rappdirs_0.3.3              uwot_0.1.8                 
##  [19] dqrng_0.2.1                 jpeg_0.1-8.1               
##  [21] zlibbioc_1.32.0             pspline_1.0-18             
##  [23] pcaMethods_1.78.0           mvtnorm_1.1-1              
##  [25] htmlwidgets_1.5.4           GlobalOptions_0.1.2        
##  [27] future_1.22.1               UpSetR_1.4.0               
##  [29] laeken_0.5.2                leiden_0.3.3               
##  [31] clustree_0.4.3              parallel_3.6.3             
##  [33] scater_1.14.6               irlba_2.3.3                
##  [35] DEoptimR_1.0-9              tidygraph_1.1.2            
##  [37] Rcpp_1.0.9                  readr_2.0.2                
##  [39] KernSmooth_2.23-17          carrier_0.1.0              
##  [41] promises_1.1.0              gdata_2.18.0               
##  [43] DelayedArray_0.12.3         limma_3.42.2               
##  [45] graph_1.64.0                RcppParallel_5.1.4         
##  [47] Hmisc_4.4-0                 fs_1.5.2                   
##  [49] RSpectra_0.16-0             fastmatch_1.1-0            
##  [51] ranger_0.12.1               digest_0.6.25              
##  [53] png_0.1-7                   sctransform_0.2.1          
##  [55] cowplot_1.0.0               DOSE_3.12.0                
##  [57] here_1.0.1                  TInGa_0.0.0.9000           
##  [59] ggraph_2.0.3                pkgconfig_2.0.3            
##  [61] GO.db_3.10.0                DelayedMatrixStats_1.8.0   
##  [63] gower_0.2.1                 ggbeeswarm_0.6.0           
##  [65] iterators_1.0.12            DropletUtils_1.6.1         
##  [67] reticulate_1.26             clusterProfiler_3.14.3     
##  [69] SummarizedExperiment_1.16.1 circlize_0.4.15            
##  [71] beeswarm_0.4.0              GetoptLong_1.0.5           
##  [73] xfun_0.35                   bslib_0.3.1                
##  [75] zoo_1.8-10                  tidyselect_1.1.0           
##  [77] reshape2_1.4.4              purrr_0.3.4                
##  [79] ica_1.0-2                   pcaPP_1.9-73               
##  [81] viridisLite_0.3.0           rtracklayer_1.46.0         
##  [83] rlang_1.0.2                 hexbin_1.28.1              
##  [85] jquerylib_0.1.4             dyneval_0.9.9              
##  [87] glue_1.4.2                  RColorBrewer_1.1-2         
##  [89] matrixStats_0.56.0          stringr_1.4.0              
##  [91] lava_1.6.7                  europepmc_0.3              
##  [93] DESeq2_1.26.0               recipes_0.1.17             
##  [95] labeling_0.3                harmony_0.1.0              
##  [97] httpuv_1.5.2                class_7.3-17               
##  [99] BiocNeighbors_1.4.2         DO.db_2.9                  
## [101] annotate_1.64.0             jsonlite_1.7.2             
## [103] XVector_0.26.0              bit_4.0.4                  
## [105] mime_0.9                    aquarius_0.1.5             
## [107] Rsamtools_2.2.3             gridExtra_2.3              
## [109] gplots_3.0.3                stringi_1.4.6              
## [111] processx_3.5.2              gsl_2.1-6                  
## [113] bitops_1.0-6                cli_3.0.1                  
## [115] batchelor_1.2.4             RSQLite_2.2.0              
## [117] randomForest_4.6-14         tidyr_1.1.4                
## [119] data.table_1.14.2           rstudioapi_0.13            
## [121] org.Mm.eg.db_3.10.0         GenomicAlignments_1.22.1   
## [123] nlme_3.1-147                qvalue_2.18.0              
## [125] scran_1.14.6                locfit_1.5-9.4             
## [127] scDblFinder_1.1.8           listenv_0.8.0              
## [129] ggthemes_4.2.4              gridGraphics_0.5-0         
## [131] R.oo_1.24.0                 dbplyr_1.4.4               
## [133] BiocGenerics_0.32.0         TTR_0.24.2                 
## [135] readxl_1.3.1                lifecycle_1.0.1            
## [137] timeDate_3043.102           ggpattern_0.3.1            
## [139] munsell_0.5.0               cellranger_1.1.0           
## [141] R.methodsS3_1.8.1           proxyC_0.1.5               
## [143] visNetwork_2.0.9            caTools_1.18.0             
## [145] codetools_0.2-16            Biobase_2.46.0             
## [147] GenomeInfoDb_1.22.1         vipor_0.4.5                
## [149] lmtest_0.9-38               msigdbr_7.5.1              
## [151] htmlTable_1.13.3            triebeard_0.3.0            
## [153] lsei_1.2-0                  xtable_1.8-4               
## [155] ROCR_1.0-7                  BiocManager_1.30.10        
## [157] scatterplot3d_0.3-41        abind_1.4-5                
## [159] farver_2.0.3                parallelly_1.28.1          
## [161] RANN_2.6.1                  askpass_1.1                
## [163] GenomicRanges_1.38.0        RcppAnnoy_0.0.16           
## [165] tibble_3.1.5                ggdendro_0.1-20            
## [167] cluster_2.1.0               future.apply_1.5.0         
## [169] Seurat_3.1.5                dendextend_1.15.1          
## [171] Matrix_1.3-2                ellipsis_0.3.2             
## [173] prettyunits_1.1.1           lubridate_1.7.9            
## [175] ggridges_0.5.2              igraph_1.2.5               
## [177] RcppEigen_0.3.3.7.0         fgsea_1.12.0               
## [179] remotes_2.4.2               scBFA_1.0.0                
## [181] destiny_3.0.1               VIM_6.1.1                  
## [183] testthat_3.1.0              htmltools_0.5.2            
## [185] BiocFileCache_1.10.2        yaml_2.2.1                 
## [187] utf8_1.1.4                  plotly_4.9.2.1             
## [189] XML_3.99-0.3                ModelMetrics_1.2.2.2       
## [191] e1071_1.7-3                 foreign_0.8-76             
## [193] withr_2.5.0                 fitdistrplus_1.0-14        
## [195] BiocParallel_1.20.1         xgboost_1.4.1.1            
## [197] bit64_4.0.5                 foreach_1.5.0              
## [199] robustbase_0.93-9           Biostrings_2.54.0          
## [201] GOSemSim_2.13.1             rsvd_1.0.3                 
## [203] memoise_2.0.0               evaluate_0.18              
## [205] forcats_0.5.0               rio_0.5.16                 
## [207] geneplotter_1.64.0          tzdb_0.1.2                 
## [209] caret_6.0-86                ps_1.6.0                   
## [211] DiagrammeR_1.0.6.1          curl_4.3                   
## [213] fdrtool_1.2.15              fansi_0.4.1                
## [215] highr_0.8                   urltools_1.7.3             
## [217] xts_0.12.1                  GSEABase_1.48.0            
## [219] acepack_1.4.1               edgeR_3.28.1               
## [221] checkmate_2.0.0             scds_1.2.0                 
## [223] cachem_1.0.6                npsurv_0.4-0               
## [225] babelgene_22.3              rjson_0.2.20               
## [227] openxlsx_4.1.5              ggrepel_0.9.1              
## [229] clue_0.3-60                 rprojroot_2.0.2            
## [231] stabledist_0.7-1            tools_3.6.3                
## [233] sass_0.4.0                  nichenetr_1.1.1            
## [235] magrittr_2.0.1              RCurl_1.98-1.2             
## [237] proxy_0.4-24                car_3.0-11                 
## [239] ape_5.3                     ggplotify_0.0.5            
## [241] xml2_1.3.2                  httr_1.4.2                 
## [243] assertthat_0.2.1            rmarkdown_2.18             
## [245] boot_1.3-25                 globals_0.14.0             
## [247] R6_2.4.1                    Rhdf5lib_1.8.0             
## [249] nnet_7.3-14                 RcppHNSW_0.2.0             
## [251] progress_1.2.2              genefilter_1.68.0          
## [253] statmod_1.4.34              gtools_3.8.2               
## [255] shape_1.4.6                 HDF5Array_1.14.4           
## [257] BiocSingular_1.2.2          rhdf5_2.30.1               
## [259] splines_3.6.3               AUCell_1.8.0               
## [261] carData_3.0-4               colorspace_1.4-1           
## [263] generics_0.1.0              stats4_3.6.3               
## [265] base64enc_0.1-3             dynfeature_1.0.0           
## [267] smoother_1.1                gridtext_0.1.1             
## [269] pillar_1.6.3                tweenr_1.0.1               
## [271] sp_1.4-1                    ggplot.multistats_1.0.0    
## [273] rvcheck_0.1.8               GenomeInfoDbData_1.2.2     
## [275] plyr_1.8.6                  gtable_0.3.0               
## [277] zip_2.2.0                   knitr_1.41                 
## [279] latticeExtra_0.6-29         biomaRt_2.42.1             
## [281] IRanges_2.20.2              fastmap_1.1.0              
## [283] ADGofTest_0.3               copula_1.0-0               
## [285] doParallel_1.0.15           AnnotationDbi_1.48.0       
## [287] vcd_1.4-8                   babelwhale_1.0.1           
## [289] openssl_1.4.1               scales_1.1.1               
## [291] backports_1.2.1             S4Vectors_0.24.4           
## [293] ipred_0.9-12                enrichplot_1.6.1           
## [295] hms_1.1.1                   ggforce_0.3.1              
## [297] Rtsne_0.15                  shiny_1.7.1                
## [299] numDeriv_2016.8-1.1         polyclip_1.10-0            
## [301] lazyeval_0.2.2              Formula_1.2-3              
## [303] tsne_0.1-3                  crayon_1.3.4               
## [305] MASS_7.3-54                 pROC_1.16.2                
## [307] viridis_0.5.1               dynparam_1.0.0             
## [309] rpart_4.1-15                zinbwave_1.8.0             
## [311] compiler_3.6.3              ggtext_0.1.0